False positive results in cytotoxicity testing due to unexpectedly volatile compounds.
We investigated the cytotoxicity of different dental materials according to the study protocol adopted by our lab for the screening of new materials. Experimental parameters used in such testing are addressed mainly in documents EN 30993 "Biological evaluation of medical devices, Part 5: Tests for cytotoxicity: in vitro methods" and "Biological evaluation of medical devices, Part 12: Sample preparation and reference materials." Cells were cultured in microplates and challenged with aqueous extracts of the materials. The assay methods were neutral red- and propidium iodide-uptake assays, both indicative of cell viability and able to provide quantitative data. The observation of contrasting results for one material using the above-mentioned methods raised some concern about the assay system used. With further experimentation, it appeared that a sustained release of volatile substances still present in one extract exerted a toxic effect in neighboring cultures. It is concluded that in the microenvironment of a microplate the distribution of samples cannot be disregarded, as it may be responsible for toxicity cross-contamination. Moreover, the use of more than one single method has to be recommended in cytotoxicity testing, in order to avoid false positive results due to experimental artifacts.
Ciapetti
G
G
Laboratory for Biocompatibility Research on Implant Materials, Istituti Ortopedici Rizzoli, Bologna, Italy.
Granchi
D
D
Verri
E
E
Savarino
L
L
Stea
S
S
Savioli
F
F
Gori
A
A
Pizzoferrato
A
A
D000818:Animals; D016477:Artifacts; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents; D003764:Dental Materials / Q000633:toxicity*; D008422:Materials Testing; D051379:Mice; D009499:Neutral Red; D010969:Plastics; D011419:Propidium; D014835:Volatilization
D004396:Coloring Agents; D003764:Dental Materials; D010969:Plastics; D009499:Neutral Red; D011419:Propidium